Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research.
Pedersen, H. L., Fangel, J. U., McCleary, B., Ruzanski, C., Rydahl, M. G., Ralet, M. C., Farkas, V., Von Schantz, L., Marcus, S. E., Andersen, M.C. F., Field, R., Ohlin, M., Knox, J. P., Clausen, M. H. & Willats, W. G. T. (2012). Journal of Biological Chemistry, 287(47), 39429-39438.
Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.
A cellulose-binding module of the Trichoderma reesei β-mannanase Man5A increases the mannan-hydrolysis of complex substrates.
Hägglund, P., Eriksson, T., Collén, A., Nerinckx, W., Claeyssens, M. & Stålbrand, H. (2003). Journal of Biotechnology, 101(1), 37-48.
Endo-β-1,4-D-mannanases (β-mannanase; EC 126.96.36.199) are endohydrolases that participate in the degradation of hemicellulose, which is closely associated with cellulose in plant cell walls. The β-mannanase from Trichoderma reesei (Man5A) is composed of an N-terminal catalytic module and a C-terminal carbohydrate-binding module (CBM). In order to study the properties of the CBM, a construct encoding a mutant of Man5A lacking the part encoding the CBM (Man5AΔCBM), was expressed in T. reesei under the regulation of the Aspergillus nidulans gpdA promoter. The wild-type enzyme was expressed in the same way and both proteins were purified to electrophoretic homogeneity using ion-exchange chromatography. Both enzymes hydrolysed mannopentaose, soluble locust bean gum galactomannan and insoluble ivory nut mannan with similar rates. With a mannan/cellulose complex, however, the deletion mutant lacking the CBM showed a significant decrease in hydrolysis. Binding experiments using activity detection of Man5A and Man5AΔCBM suggests that the CBM binds to cellulose but not to mannan. Moreover, the binding of Man5A to cellulose was compared with that of an endoglucanase (Cel7B) from T. reesei.
Fractionation of extracted hemicellulosic saccharides from Pinus pinaster wood by multistep membrane processing.
González-Muñoz, M. J., Rivas, S., Santos, V. & Parajó, J. C. (2013). Journal of Membrane Science, 428, 281-289.
Hemicelluloses of Pinus pinaster wood were selectively separated from cellulose and lignin by reaction with hot, compressed water (autohydrolysis) under optimized conditions. The reaction liquor contained polymeric or oligomeric hemicellulose saccharides (POHS, accounting jointly for 69.6% of the dissolved wood fraction), followed by monosaccharides (accounting for 20.0% of the non-volatile compounds), and non-saccharide compounds. For concentration, purification and fractionation purposes, liquors from hydrothermal processing were subjected to consecutive steps of diafiltration and concentration using membranes of 10, 5, 3, 1 and 0.3 kDa molar mass cut-off. Samples from selected process streams were characterized by chromatographic and spectrometric methods. The experimental results provided information on the separation and refining effects achieved by the various membrane processing steps, which affect the technological properties of products.
Structure of a mannan-specific family 35 carbohydrate-binding module: evidence for significant conformational changes upon ligand binding.
Tunnicliffe, R. B., Bolam, D. N., Pell, G., Gilbert, H. J. & Williamson, M. P. (2005). Journal of Molecular Biology, 347(2), 287-296.
Enzymes that digest plant cell wall polysaccharides generally contain non-catalytic, carbohydrate-binding modules (CBMs) that function by attaching the enzyme to the substrate, potentiating catalytic activity. Here, we present the first structure of a family 35 CBM, derived from the Cellvibrio japonicus β-1,4-mannanase Man5C. The NMR structure has been determined for both the free protein and the protein bound to mannopentaose. The data show that the protein displays a typical β-jelly-roll fold. Ligand binding is not located on the concave surface of the protein, as occurs in many CBMs that display the jelly-roll fold, but is formed by the loops that link the two β-sheets of the protein, similar to family 6 CBMs. In contrast to the majority of CBMs, which are generally rigid proteins, CBM35 undergoes significant conformational change upon ligand binding. The curvature of the binding site and the narrow binding cleft are likely to be the main determinants of binding specificity. The predicted solvent exposure of O6 at several subsites provides an explanation for the observed accommodation of decorated mannans. Two of the key aromatic residues in Man5C-CBM35 that interact with mannopentaose are conserved in mannanase-derived CBM35s, which will guide specificity predictions based on the primary sequence of proteins in this CBM family.
A (1→4)-β-mannan-specific monoclonal antibody and its use in the immunocytochemical location of galactomannans.
Pettolino, F. A., Hoogenraad, N. J., Ferguson, C., Bacic, A., Johnson, E. & Stone, B. A. (2001). Planta, 214(2), 235-242.
Galactomannan was coupled to a protein carrier for the preparation of monoclonal antibodies. The monoclonal antibodies generated bound to galactomannans from different sources as well as to glucomannan and galactoglucomannan. One monoclonal antibody, BGM C6, was characterised and found to be specific for (1→4)-β-linked mannopyranosyl residues; it had a binding affinity estimated at 1×10-6) M for the (1→4)-β-linked mannohexaose. BGM C6 was used in immunogold labelling studies to locate galactomannans in the endosperm walls of normal coconuts (Cocos nucifera L.) and those of the mutant makapuno at two different developmental stages. The pattern and intensity of antibody labelling varied for each type of coconut at the mature and immature stages, indicating differences in the galactomannan composition of the endosperm walls.
A novel thermophilic endo-β-1, 4-mannanase from Aspergillus nidulans XZ3: functional roles of carbohydrate-binding module and Thr/Ser-rich linker region.
Lu, H., Luo, H., Shi, P., Huang, H., Meng, K., Yang, P. & Yao, B. (2014). Applied Microbiology and Biotechnology, 98(5), 2155-2163.
The gene man5XZ3 from Aspergillus nidulans XZ3 encodes a multimodular β-mannanase of glycoside hydrolase family 5 that consists of a family 1 carbohydrate-binding module (CBM1), a Thr/Ser-rich linker region, and a catalytic domain. Recombinant Man5XZ3 and its two truncated derivatives, Man5ΔCBM (removing the CBM1) and Man5ΔCL (removing both the CBM1 and linker region), were produced in Pichia pastoris and showed significant variance in the secondary structure. The three enzymes had similar biochemical properties, such as optimal pH and temperature (pH 5.0 and 80°C) and excellent pH stability at pH 4.0–10.0. Removal of the CBM1 alone could improve the thermostability of Man5XZ3, but further removal of the linker region resulted in worse thermostability. Man5XZ3 retained greater enzyme activity in the presence of an organic solvent (acetone), two detergents (SDS and Triton X-100), and a chaotropic agent (urea) compared with Man5ΔCBM and Man5ΔCL. This study provides an excellent β-mannanase candidate favorable for various industries and primarily demonstrates the relationship between enzyme structure and function.
LeMAN4 endo-β-mannanase from ripe tomato fruit can act as a mannan transglycosylase or hydrolase.
Schröder, R., Wegrzyn, T. F., Sharma, N. N. & Atkinson, R. G. (2006). Planta, 224(5), 1091-1102.
Mannan transglycosylases are cell wall enzymes able to transfer part of the mannan polysaccharide backbone to mannan-derived oligosaccharides (Schröder et al. in Planta 219:590–600, 2004). Mannan transglycosylase activity was purified to near homogeneity from ripe tomato fruit. N-terminal sequencing showed that the dominant band seen on SDS-PAGE was identical to LeMAN4a, a hydrolytic endo-β-mannanase found in ripe tomato fruit (Bewley et al. in J Exp Bot 51:529–538, 2000). Recombinant LeMAN4a protein expressed in Escherichia coli exhibited both mannan hydrolase and mannan transglycosylase activity. Western analysis of ripe tomato fruit tissue using an antibody raised against tomato seed endo-β-mannanase revealed four isoforms present after 2D-gel electrophoresis in the pH range 6–11. On separation by preparative liquid isoelectric focussing, these native isoforms exhibited different preferences for transglycosylation and hydrolysis. These results demonstrate that endo-β-mannanase has two activities: it can either hydrolyse mannan polysaccharides, or in the presence of mannan-derived oligosaccharides, carry out a transglycosylation reaction. We therefore propose that endo-β-mannanase should be renamed mannan transglycosylase/hydrolase, in accordance with the nomenclature established for xyloglucan endotransglucosylase/hydrolase. The role of endo-acting mannanases in modifying the structure of plant cell walls during cell expansion, seed germination and fruit ripening may need to be reinterpreted in light of their potential action as transglycosylating or hydrolysing enzymes.
Substrate Specificities of α-Galactosidase from Rice.
Li, S. H., Zhu, M. P. & Li, T. P. (2011). Advanced Materials Research, 183, 447-451.
The α-galactosidase from rice cleaved not only α-D-galactosyl residues from the non-reducing end of substrates such as melibiose, raffinose and stachyose, but also liberated the terminal galactosyl residues attached O-6 position of the reducing-end mannosyl residue in mannobiose and mannotriose. In addition, the enzyme tore off the stubbed galactosyl residues attached inner-mannosyl residues in mannopentaose. It also could catalyze efficient degalactosylation of galactomannans, such as guar gum and locust bean gum.
The modular architecture of Cellvibrio japonicus mannanases in glycoside hydrolase families 5 and 26 points to differences in their role in mannan degradation.
Hogg, D., Pell, G., Dupree, P., Goubet, F., Martin-Orue, S., Armand, S. & Gilbert, H. (2003). Biochem. J, 371, 1027-1043.
β-1,4-Mannanases (mannanases), which hydrolyse mannans and glucomannans, are located in glycoside hydrolase families (GHs) 5 and 26. To investigate whether there are fundamental differences in the molecular architecture and biochemical properties of GH5 and GH26 mannanases, four genes encoding these enzymes were isolated from Cellvibrio japonicus and the encoded glycoside hydrolases were characterized. The four genes, man5A, man5B, man5C and man26B, encode the mannanases Man5A, Man5B, Man5C and Man26B, respectively. Man26B consists of an N-terminal signal peptide linked via an extended serine-rich region to a GH26 catalytic domain. Man5A, Man5B and Man5C contain GH5 catalytic domains and non-catalytic carbohydrate-binding modules (CBMs) belonging to families 2a, 5 and 10; Man5C in addition contains a module defined as X4 of unknown function. The family 10 and 2a CBMs bound to crystalline cellulose and ivory nut crystalline mannan, displaying very similar properties to the corresponding family 10 and 2a CBMs from Cellvibrio cellulases and xylanases. CBM5 bound weakly to these crystalline polysaccharides. The catalytic domains of Man5A, Man5B and Man26B hydrolysed galactomannan and glucomannan, but displayed no activity against crystalline mannan or cellulosic substrates. Although Man5C was less active against glucomannan and galactomannan than the other mannanases, it did attack crystalline ivory nut mannan. All the enzymes exhibited classic endo-activity producing a mixture of oligosaccharides during the initial phase of the reaction, although their mode of action against manno-oligosaccharides and glucomannan indicated differences in the topology of the respective substrate-binding sites. This report points to a different role for GH5 and GH26 mannanases from C. japonicus. We propose that as the GH5 enzymes contain CBMs that bind crystalline polysaccharides, these enzymes are likely to target mannans that are integral to the plant cell wall, while GH26 mannanases, which lack CBMs and rapidly release mannose from polysaccharides and oligosaccharides, target the storage polysaccharide galactomannan and manno-oligosaccharides.
Model for random hydrolysis and end degradation of linear polysaccharides: Application to the thermal treatment of mannan in solution.
Nattorp, A., Graf, M., Spühler, C. & Renken, A. (1999). Industrial & Engineering Chemistry Research, 38(8), 2919-2926.
The kinetics for homogeneous hydrolysis of mannan is studied in a batch reactor at temperatures from 160 to 220°C. A formate buffer ensures a pH of 3.8−4.0, measured at 25°C. Samples are analyzed for oligosaccharides up to a degree of polymerization of 6 and also for the total amount of mannose after acid hydrolysis. A mathematical model with two reactions (1, random hydrolysis of the glucosidic bonds; 2, degradation of the reducing end of the molecule) describes accurately the time course of oligosaccharides. Optimized rate constants follow closely an Arrhenius relationship, with the degradation having a higher activation energy (140 kJ/mol) than the hydrolysis (113 kJ/mol). The mathematical model has the advantage that production of small molecules is independent of the initial chain-length distribution as long as the average initial chain length is some 5 times longer than the largest species measured. It can be applied to first-order depolymerization of other linear polymers with one link type in order to determine reaction rate constants or make predictions about molecular weight distribution on the base of known reaction rate constants.
Acidic β-mannanase from Penicillium pinophilum C1: Cloning, characterization and assessment of its potential for animal feed application.
Cai, H., Shi, P., Luo, H., Bai, Y., Huang, H., Yang, P. & Yao, B. (2011). Journal of Bioscience and Bioengineering, 112(6), 551-557.
The β-mannanase gene, man5C1, was cloned from Penicillium pinophilum C1, a strain isolated from the acidic wastewater of a tin mine in Yunnan, China, and expressed in Pichia pastoris. The sequence analysis displayed the gene consists of a 1221-bp open reading frame encoding a protein of 406 amino acids (Man5C1). The deduced amino acid sequence of Man5C1 showed the highest homology of 57.8% (identity) with a characterized β-mannanase from Aspergillus aculeatus belonging to glycoside hydrolase family 5. The purified rMan5C1 had a high specific activity of 1035 U mg-1 towards locust bean gum (LBG) and showed highest activity at pH 4.0 and 70°C. rMan5C1 was adaptable to a wide range of acidity, retaining > 60% of its maximum activity at pH 3.0–7.0. The enzyme was stable over a broad pH range (3.0 to 10.0) and exhibited good thermostability at 50°C. The Km and Vmax values were 5.6 and 4.8 mg mL-1, and 2785 and 1608 μmol min-1 mg-1, respectively, when LBG and konjac flour were used as substrates. The enzyme had strong resistance to most metal ions and proteases (pepsin and trypsin), and released 8.96 mg g-1 reducing sugars from LBG in the simulated gastric fluid. All these favorable properties make rMan5C1 a promising candidate for use in animal feed.